A potential model system for screening of methods purported to remove or disinfectant TSE agents is in initial stages of development in collaboration between LMD and the Laboratory of Analytical Chemistry, DMPQ, CBER, attempting to confirm reports that PC12 rat-pheochromocytoma cells infected with some strains of the scrapie agent undergo marked reduction in acetyl-cholinesterase neurotransmitter activity while maintaining normal levels of adrenergic activity. Should that pilot study succeed, PC12 cells might provide a suitable simplified assay to detect scrapie agent as a preliminary screening test of disinfectant and sterilization methods as described above. Methods that fail to remove infectivity of scrapie agent detectable in cell culture would clearly be inadequate for practical use--where infecting doses of agent are potentially much higher than those detected by cell cultures--and would not merit further investigation in rodents. In FY 98, BSL-2 laboratory facilities and procedures suitable for the safe study of the infectious agents in cell cultures (using BSL-2 equipment and practices and elevated autoclave temperatures) were established, and basic cell-culture and HPLC-based enzymological methods needed for the model were successfully developed. In FY 99, data from PC-12 cell cultures infected with scrapie brain analyzed by HPLC demonstrated a decrease in acetycholinesterase activity compared to activity in mock-infected cells. Further studies are in progress to optimize enzyme release from harvested PC-12 cells and to determine if the reduction in the level of acetylcholinesterase in scrapie infected cells compared to that in mock-infected cells is significant, consistent and reproducible. In addition, in collaboration with Thomas Flynn, Ph.D., CFSAN, and Andrew Scallet, Ph.D., NCTR, an in vitro study using primary reaggregated rat retinal cell cultures has been initiated. Such cultures exhibit histotypic differentiation. Neurochemical studies have shown that the differentiated cells express the neurotransmitter enzymes that may be inhibited by scrapie and thus may provide an alternatative means of detecting scrapie infection. Recently, using PCR techniques, we have begun to examine the mutation rate in the open reading frame of the gene encoding the prion protein (PRNP gene) in a continuous cell line under consideration as a vaccine substrate. (Mutations in the PRNP gene have been associated with familial TSEs.) The study will examine changes in the PRNP gene sequences in reference HeLa cells, derived from a cervical carcinoma in 1951, and in six cell lines contaminated with HeLa cells during the 1950's and early 1960's. The lines we propose to study, therefore, have undergone numerous serial passages in different laboratories during more than 30 years, suggesting that any genetic instability in the PRNP gene might be detected.